The work presented here has a multidisciplinary character, having as a common factor the characterization of self-assembled nanostructures through force spectroscopy. Exploring AFM as a tool for characterizing self-assembly and interaction forces in soft matter nanostructures, three different Bio and nonbiological systems where investigated, all of them share the common characteristic of being soft matter molecular structures at the nanoscale. The studied systems in question are: a) Polyelectrolyte – lipid nanocomposites. Single polyelectrolyte adsorption-desorption from supported lipid bilayers, b) Polyelectrolyte brushes and c) Virus-Like particles (VLPs). The scientific interest and industrial applications for each of these different nanostructures is broad, and their potential uses in the near future ranges from smart nanocontainers for drug and gene delivery, surface platforms for molecular recognition to the development of new nanodevices with ultrasensitive external stimuli responsiveness. These nano-structures are constructed following assembly of smaller subunits and belong to representative examples of soft matter in modern nanotechnology. The stability, behavior, properties and long term durability of these self-organized structures depends strongly on the environmental conditions to which they are exposed since their building mechanism is a balance between attractive noncovalent interactions and momentum transmitted collisions due Brownian motion of the solvent molecules. For example a set of long chain molecules firmly attached to one end to a surface will alter their conformation as the space between them is reduced or the environmental conditions are modified (i.e. ionic strength, pH or temperature). For a highly packed condition, this fuzzy surface known as a polyelectrolyte brush will then behave as a responsive material with tunable responsiveness. Thus the objective in the present case was to investigate the change in morphology and the mechanical response of a polyelectrolyte brush to external forces by application of AFM nanoindentations under different ionic strength conditions. The degree of penetration of the AFM tip through the brush will provide insights into the forces exerted by the brush against the tip. Compressions on the brush should aid to characterize its changes in compressibility for different salt concentrations. For the second chosen system, the interaction between two assembled interfaces was investigated at the single molecular level. A multilayered film formed by the consecutive assembly of oppositely charged polyelectrolytes and subsequently coated with a lipid membrane represents a fascinating soft composite material resembling more than a few structural components emerging in living organisms. The fluid bilayer, thus provide a biocompatible interface where additional functionalities can further be integrated (fusion peptides for instance). The smooth polymer cushion confers not only structural flexibility but also adaptability of the chosen substrate properties to be coated. This type of interface could be useful in the development of novel molecular biosensors with single molecule recognition capacities or in the fabrication of assays against pathogenic agents. The aim of this project was to study the molecular binding mechanism between the last polyelectrolyte layer and the lipid head group of the lower lipid leaflet. Understanding this adsorption mechanism between both interfaces, should likewise contribute to improve the fabrication of lipid coated polymeric nano/micro capsules with targeting properties. For example this could be critical in the field of nonviral gene therapy, where the improvement in the design of condensates of nucleic acids and other polymers with lipids (lipoplexes) are of main interest for its posterior use as delivery vectors. Finally, viral capsids were investigated. These naturally occurring assembled nanocontainers within living organisms stand for a remarkable example of nature’s morphological designs. These structures self-assemble from a small number of different proteins occurring in identical copies. The capsid as a self-assembled structure carries multiple functions: compaction of the genome, protection against external chemical threats, target recognition, structural support and finally facilitating the release of the genome into the host cell. It is highly interesting how these different functions are organized within the capsid which consists, for example, in the case of the norovirus of 180 identical copies of one single protein. Therefore, the mechanical stability and elastic properties of virus-like particles of Rubella and Norovirus were investigated by external application of loading forces with an AFM tip. The measurements were performed under conditions relevant for the virus infection mechanism. The applied compressions on these protein shells at pH values mimicking the virus life cycle will aid to learn about possible internal transitions among proteins which may be important for switching between the various functions of the capsid. The choice of two unrelated viral systems with different entry pathways into the cell and with different morphological architectures is expected to reveal crucial information about the stability and mechanical resistance to deformation of these empty membrane-coated and bare viral capsids. This last might provide clues on the stage of particle disassembly and cargo release during the final step of the infection process.

The abundant cellular protein Cyclophilin A (CypA) was found to bind to HIV-1 capsid (CA) in 1993. Since that time, several complementary methods, including disruption of the binding interface by cyclosporine A, CA mutants, and CypA mutants, have been used to demonstrate that CypA acts within human target cells to promote HIV-1 infection. In contrast, in cells from non-human primates, CypA in target cells decreases HIV-1 infectivity, and it does so by promoting TRIM5α-mediated restriction. Using human cancer cell lines and the genetic methods available at the time, attempts to obtain evidence that CypA inhibits HIV-1 restriction by the human TRIM5α ortholog, let alone that human TRIM5α restricts HIV-1, were unsuccessful. Here we revisit the question of the mechanism by which CypA increases HIV-1 infectivity by exploiting lentiviral vectors optimized for primary human blood cells that serve as HIV-1 targets. Disruption of CA−CypA interaction is demonstrated to render HIV-1 vulnerable to endogenous human TRIM5α-mediated recognition and restriction, which occur prior to completion of reverse transcription. Identical findings were acquired with single-cycle vectors or with replication-competent viruses. Consistently, a previously identified, cyclosporine-resistant CA mutation A92E is also shown to confer resistance against restriction by human TRIM5α. Therefore, the results presented in this thesis reveal that HIV-1 exploits a host protein CypA bound to its CA to evade potent restriction by human TRIM5α. This finding not only answers a long-standing question regarding the role of CypA in HIV-1 infection, but also may reinvigorate the development of CypA inhibitors for treatment of HIV-1.

Le gène L1 encode pour la protéine majeure de la capside des papillomavirus humains (VPH). L’information relative au polymorphisme de L1 pour les types autres que VPH- 16 est jusqu’ici limitée. Cet ouvrage explore le polymorphisme de L1 en comparant les séquences des types phylogénétiquement apparentés VPH-31, -33, -35 à VPH-16. Des spécimens génitaux recueillis de 732 femmes VIH-séropositives et 323 VIHséronégatives ont été criblés à le recherche d’ADN de VPH par PCR consensus au niveau du gène L1. Les échantillons positifs pour VPH-16 (n=74), -31 (n=74), -33 (n=37) et -35 (n=58) étaient analysés par PCR-séquençage pour la totalité du gène L1. Le nombre de nucléotides substitués pour L1 variait de 19 pour VPH-33 à 52 pour VPH-31. Le rapport du nombre de variantes sur le nombre d’isolats testés était plus élevé pour VPH-31 (56.4%, p=0.05) et VPH-35 (60.3%, p=0.04) comparativement à VPH-16 (40.5%), alors que ce ratio était inférieur pour VPH-33 mais sans différence statistiquement significative (24.3%, p=0.14). La distance entre les variantes était plus grande à l’intérieur des cinq boucles présumément exposées à la surface de la protéine L1 que dans la séquence à l’extérieur (p<0.01) Des variations synonymes étaient observées chez 1.7% (95% CI 1.1- 2.3) des nucléotides intra-boucles et 2.4% (95% CI 1.2-3.7) de ceux extra-boucles. Les variations non-synonymes étaient rencontrées pour 1.8% (95% CI 1.1-2.5) des nucléotides intra-boucles et 0.2% (95% CI 0-0.4) pour les nucléotides extra-boucles. Les ratios dN/dS étaient inférieurs à 1.0 pour les régions extra-boucles et encore davantage pour les régions intra-boucles. Ces résultats suggèrent que les séquences des régions hypervariables de L1 ont été sélectionnées positivement. / The L1 gene encodes for the major capsid protein of human papillomaviruses (HPV). There is limited information on the polymorphism of L1 for types related to HPV-16. This report explores the polymorphism of L1 in phylogenetically-related types 31, 33, and 35 compared to HPV-16. Genital specimens collected from 732 HIV-seropositive and 323 HIV-seronegative women were screened for HPV DNA with consensus L1 PCR. Cervical samples positive for HPV-16 (n=74), -31 (n=78), -33 (n=37), and -35 (n=58) were further characterized by PCR-sequencing of the complete L1 gene. The number of nucleotide substitutions within L1 ranged from 19 for HPV-33 to 52 for HPV-31. The ratio of the number of variants/number of isolates tested was higher for HPV31 (56.4%, p=0.05) and HPV-35 (60.3%, p=0.04) compared to HPV-16 (40.5%), while this ratio was lower for HPV-33 (24.3%), although not significantly (p=0.14). The maximal distance between HPV variants was greater in the five putative surface-exposed loops of L1 than in sequences outside the loops (p<0.01). Synonymous variations were encountered in 1.7% (95% CI 1.1-2.3) of nucleotides inside the L1 loops and 2.4% (95% CI 1.2-3.7) of nucleotides outside the L1 loops. Non-synonymous variations were encountered in 1.8% (95% CI 1.1-2.5) of nucleotides within the L1 loops and 0.2% (95% CI 0-0.4) of nucleotides outside the loops. dN/dS ratios were below 1.0 in extra-loop and intra-loop regions, but they were lower in extra-loop regions. These results suggest that sequences within and outside the hypervariable loops of L1 were under selective constraint.

Murine polyomavirus (MPyV) is a member of the Polyomaviridae family. Its capsid is composed of the major capsid protein, VP1, and the minor proteins, VP2 and VP3. The minor capsid proteins probably assure delivery of the viral genome through the endoplasmic reticulum membrane to the nucleus during early phase of infection. However, precise mechanism is not known. Expression plasmids encoding mutated VP2 or VP3 fused with EGFP have been constructed to study the interaction of VP2 and VP3 with membranes. The mutated proteins have deletions in the predicted hydrophobic domains. In this thesis, cell localisation of mutated proteins was followed. The study revealed that the hydrophobic domain 2 is the most important for association of VP2 and VP3 with membranes, while domains 1 and 3 are rather expendable. Further, nature of VP2 and VP3 isoforms has been studied. Isoforms with different electrophoretic mobility were separated on SDS-PAGE. Consequent mass spectrometry analysis showed that they differ in deamidation of asparagine, present at both minor proteins (position 253 of VP2 and 137 of VP3). Previously, acetylation of VP3 N-terminal alanine has been identified. To elucidate the function of these modifications, mutated viruses were constructed with substitution of these amino acids. Pilot...

Le nombre croissant de nanostructures physiques et biologiques sont caractérisées par l'ordre non-cristallin et par les propriétés physiques et biologiques non-conventionnels. Parmi ses systèmes il faut distinguer les capsides virales. Ces coquilles solides qui sont formées par un certain nombre dec opies de la même protéine protègent le virus des agressions et facilitent le processus d'infection de la cellule hôte. La distribution des positions de protéines dans une capside est très régulière et montre un degré très élevé d'ordre, aussi bien orientationnel que positionnel. Les capsides virales de topologie sphérique possèdent la symétrie icosaédrique compatible avec l'ordre cristallin local, mais incompatible avec la symétrie cristalline globale et interdite dans les structures périodiques.Ici, sur l'exemple des Papovavirus, nous montrons l'existence d'un nouveau type d'organisation qui résulte dans l'ordre quasicristallin pentagonal chiral de protéines dans des capsides de topologie sphérique et géométrie dodécaédrique. La formation de cet ordre est décrite dans le cadre de la théorie de Landau de cristallisation. Les particularités de la structure sont élucidées grâce à la théorie d'élasticité des quasicristaux comme le résultat de la déformation phason nonlinéaire.La généralisation de la théorie de Landau de cristallisation que nous proposons permet également de décrire des structures quasicristallines octogonales et décagonales grâce à la minimisation contrainte de l'énergie libre, et donne un nouveau sens physique à la notion de « fenêtre de projection » utilisée dans la cristallographie multidimensionnelle. / A growing number of physical and biological nanostructures are characterized by non-crystallineorder and by unconventional physical and biological properties. Among these systems one can distinguish viral capsids. These solid shells formed by a certain number of copies of the same protein protect viruses from aggressions and facilitate infection of the host cell. Protein distributionin a capsid is quite regular and shows high degree of order, both orientational and positional. Viral capsids with spherical topology have icosahedral symmetry compatible with local crystalline orderbut incompatible with the global one and forbidden in periodic structures.Here, on the example of Papovaviruses we show the existence of a new type of organization whichresults in the chiral pentagonal quasicrystalline order of proteins in capsids with spherical topology and dodecahedral geometry. The formation of this order is described in the frame of the Landau theory of crystallization. The theory of elasticity of quasicrystals is used to show that the structure peculiarities result from the non-linear phason strain.Generalization of the proposed Landau theory of crystallization allows us to describe octagonal and decagonal quasicrystalline structures using constrained minimization of the free energy, thus giving a new physical sense to the « projection window » notion used in multi-dimensionalcrystallography.

Vacinação contra doenças virais e bacterianas tem sido uma das histórias de sucesso da medicina humana e veterinária. Vacinas genéticas são constituídas apenas de DNA (como plasmídeos) ou RNA (como mRNA), que são entregues às células alvo. Esta abordagem proporciona uma vantagem significativa, pois essas preparações em geral apresentam facilidade de construção genética, baixo custo, rapidez de produção em massa, estabilidade elevada e um perfil de segurança atraente. Vacinas baseadas em RNA recentemente receberam maior atenção porque, ao contrário de vacinas de DNA, são processadas no citoplasma, excluindo a necessidade de entrada no núcleo celular. O vírus Semliki Forest (SFV, Alphavirus, Togaviridae ) possui um RNA genômico, auto replicativo, de simples fita e polaridade positiva, protegido em uma nucleocápside icosaédrica, composta pelo próprio RNA e pela proteína de cápside viral (C), de 267 aminoácidos. A nucleocápside contém 180 cópias da proteína C e é coberta por uma membrana lipoprotéica contendo as glicoproteínas virais. Para realizar a entrega de moléculas de RNA com fins de imunização é fundamental que o material esteja protegido da ação de enzimas. Assim, o objetivo deste trabalho foi expressar a proteína C recombinante de SFV em E.coli , associá-la in vitro com RNA auto replicativo, contendo gene repórter e fazer entrega deste complexo à célula alvo. Para isso, o gene da proteína C foi obtido de forma sintética e clonado em um vetor de expressão em bactérias, pET-28a(&#43;). Para a expressão da proteína C recombinante foram utilizadas bactérias E. coli BL21(DE3) e testadas diferentes temperaturas após indução com IPTG. A proteína obtida foi purificada por cromatografia de afinidade e a amostra foi dialisada utilizando coluna de dessalinização. Após a purificação, a proteína recombinante foi submetida a uma associação in vitro com RNA, obtido através de transcrição in vitro . A eficiência de formação do complexo Proteína-RNA foi medida pela quantidade diferencial de RNA livre antes e após o procedimento por eletroforese em gel de agarose e pela análise através de imagens obtidas por microscopia eletrônica de transmissão. A entrega do complexo Proteína-RNA foi realizada de forma eficiente à célula alvo. Desta forma, de maneira geral, a proteína de capsídeo do vírus SFV é capaz de se associar in vitro com RNA auto replicativo, trazendo proteção e estabilidade ao mesmo, bem como realizar a entrega desse material à célula alvo, através da desmontagem da nucleocápside no interior da célula hospedeira. / Vaccination against viral and bacterial diseases has been one of the success stories of human and veterinary medicine. Genetic vaccines consist only of DNA (such as plasmids) or RNA (such as mRNA), which are delivered to target cells. This approach provides a significant advantage as these preparations generally have ease of genetic engineering, low cost, fast mass production, high stability and attractive safety profile. Unlike DNA vaccines, RNAbased vaccines are processed into the cytoplasm, excluding the need for entry into the cell nucleus. The Semliki Forest virus (SFV, Alphavirus, Togaviridae ) has a positive-strand and auto amplify RNA, protected in an icosahedral nucleocapsid, composed of viral RNA and the 267 amino acid capsid protein (C). The nucleocapsid contains 180 copies of protein C and is covered by a lipoprotein membrane containing the viral glycoproteins. In order to carry out the delivery of RNA molecules for immunization purposes, it is essential that the material is protected from the action of enzymes. Thus, the objective of this work was to express recombinant SFV C protein in E.coli, to associate it in vitro with auto replicative RNA, containing reporter gene and to deliver this complex to the target cell. For this, the protein C gene was synthesized and cloned into a bacterial expression vector, pET-28a (&#43;). For expression of recombinant protein C, E. coli BL21 (DE3) bacteria were used and different temperatures tested after IPTG induction. The obtained protein was purified by affinity chromatography and the sample was dialyzed using desalting column. After purification, the recombinant protein was subjected to an in vitro association with RNA, obtained by in vitro transcription. The efficiency of formation of the Protein-RNA complex was measured by the differential amount of free RNA before and after the procedure by agarose gel electrophoresis and by the analysis through images obtained by transmission electron microscopy. Delivery of the Protein-RNA complex was efficiently performed on the target cell. Thus, in general, the SFV virus capsid protein is able to associate in vitro with auto amplify RNA, providing protection and stability to it, as well as delivering this material to the target cell, by disassembling the nucleocapsid in the interior of the host cell.

Knowledge about structure and dynamics of the important biological macromolecules — proteins, nucleic acids, lipids and sugars — helps to understand their function. Atomic-resolution structures of macromolecules are routinely captured with X-ray crystallography and other techniques. In this thesis, simulations are used to explore the dynamics of the molecules beyond the static structures. Viruses are machines constructed from macromolecules. Crystal structures of them reveal little to no information about their genomes. In simulations of empty capsids, we observed a correlation between the spatial distribution of chloride ions in the solution and the position of RNA in crystals of satellite tobacco necrosis virus (STNV) and satellite tobacco mosaic virus (STMV). In this manner, structural features of the non-symmetric RNA could also be inferred. The capsid of STNV binds calcium ions on the icosahedral symmetry axes. The release of these ions controls the activation of the virus particle upon infection. Our simulations reproduced the swelling of the capsid upon removal of the ions and we quantified the water permeability of the capsid. The structure and dynamics of the expanded capsid suggest that the disassembly is initiated at the 3-fold symmetry axis. Several experimental methods require biomolecular samples to be injected into vacuum, such as mass-spectrometry and diffractive imaging of single particles. It is therefore important to understand how proteins and molecule-complexes respond to being aerosolized. In simulations we mimicked the dehydration process upon going from solution into the gas phase. We find that two important factors for structural stability of proteins are the temperature and the level of residual hydration. The simulations support experimental claims that membrane proteins can be protected by a lipid micelle and that a non-membrane protein could be stabilized in a reverse micelle in the gas phase. A water-layer around virus particles would impede the signal in diffractive experiments, but our calculations estimate that it should be possible to determine the orientation of the particle in individual images, which is a prerequisite for three-dimensional reconstruction. / BMC B41, 25/5, 9:15

The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Title from PDF of title page (University of Missouri--Columbia, viewed on March 10, 2010). Vita. Thesis advisor: David Pintel "August 2008" Includes bibliographical references

Le gène L1 encode pour la protéine majeure de la capside des papillomavirus humains (VPH). L’information relative au polymorphisme de L1 pour les types autres que VPH- 16 est jusqu’ici limitée. Cet ouvrage explore le polymorphisme de L1 en comparant les séquences des types phylogénétiquement apparentés VPH-31, -33, -35 à VPH-16. Des spécimens génitaux recueillis de 732 femmes VIH-séropositives et 323 VIHséronégatives ont été criblés à le recherche d’ADN de VPH par PCR consensus au niveau du gène L1. Les échantillons positifs pour VPH-16 (n=74), -31 (n=74), -33 (n=37) et -35 (n=58) étaient analysés par PCR-séquençage pour la totalité du gène L1. Le nombre de nucléotides substitués pour L1 variait de 19 pour VPH-33 à 52 pour VPH-31. Le rapport du nombre de variantes sur le nombre d’isolats testés était plus élevé pour VPH-31 (56.4%, p=0.05) et VPH-35 (60.3%, p=0.04) comparativement à VPH-16 (40.5%), alors que ce ratio était inférieur pour VPH-33 mais sans différence statistiquement significative (24.3%, p=0.14). La distance entre les variantes était plus grande à l’intérieur des cinq boucles présumément exposées à la surface de la protéine L1 que dans la séquence à l’extérieur (p<0.01) Des variations synonymes étaient observées chez 1.7% (95% CI 1.1- 2.3) des nucléotides intra-boucles et 2.4% (95% CI 1.2-3.7) de ceux extra-boucles. Les variations non-synonymes étaient rencontrées pour 1.8% (95% CI 1.1-2.5) des nucléotides intra-boucles et 0.2% (95% CI 0-0.4) pour les nucléotides extra-boucles. Les ratios dN/dS étaient inférieurs à 1.0 pour les régions extra-boucles et encore davantage pour les régions intra-boucles. Ces résultats suggèrent que les séquences des régions hypervariables de L1 ont été sélectionnées positivement. / The L1 gene encodes for the major capsid protein of human papillomaviruses (HPV). There is limited information on the polymorphism of L1 for types related to HPV-16. This report explores the polymorphism of L1 in phylogenetically-related types 31, 33, and 35 compared to HPV-16. Genital specimens collected from 732 HIV-seropositive and 323 HIV-seronegative women were screened for HPV DNA with consensus L1 PCR. Cervical samples positive for HPV-16 (n=74), -31 (n=78), -33 (n=37), and -35 (n=58) were further characterized by PCR-sequencing of the complete L1 gene. The number of nucleotide substitutions within L1 ranged from 19 for HPV-33 to 52 for HPV-31. The ratio of the number of variants/number of isolates tested was higher for HPV31 (56.4%, p=0.05) and HPV-35 (60.3%, p=0.04) compared to HPV-16 (40.5%), while this ratio was lower for HPV-33 (24.3%), although not significantly (p=0.14). The maximal distance between HPV variants was greater in the five putative surface-exposed loops of L1 than in sequences outside the loops (p<0.01). Synonymous variations were encountered in 1.7% (95% CI 1.1-2.3) of nucleotides inside the L1 loops and 2.4% (95% CI 1.2-3.7) of nucleotides outside the L1 loops. Non-synonymous variations were encountered in 1.8% (95% CI 1.1-2.5) of nucleotides within the L1 loops and 0.2% (95% CI 0-0.4) of nucleotides outside the loops. dN/dS ratios were below 1.0 in extra-loop and intra-loop regions, but they were lower in extra-loop regions. These results suggest that sequences within and outside the hypervariable loops of L1 were under selective constraint.

To initiate algal virus research in the Bay of Quinte, three stations were sampled biweekly throughout 2011. By targeting algal virus DNA polymerase, major capsid protein genes (MCP), and a Microcystis aeruginosa cyanophage (Ma-LMM01) tail sheath protein gene, PCR amplification revealed diverse and unique Phycodnaviruses (viruses of eukaryotic algae) and cyanophage. When analysed statistically, patterns of virus abundance suggested that the seasonality of any one virus cannot be generalised to predict that of other viruses, even among closely related viruses. This study also demonstrated a strong relationship between algal virus abundance and host biomass. It was found that despite the apparent heterogeneity of virus abundance across the Bay, virus abundance patterns clustered by sampling date and geographic location. By providing evidence for diverse algal viruses with complex seasonality, this work highlights significant gaps in the current understanding of Bay of Quinte phytoplankton ecology.